Conformational Lock Research Articles

Calmodulin-dependent regulation of mammalian nitric oxide synthase.

The nitric oxide synthases are large, modular, dimeric enzymes composed of a reductase domain, which is related to cytochrome P450 reductase, and a structurally unique oxygenase domain containing a Cys-ligated haem. Both the neuronal and endothelial isoforms are activated by the reversible binding of calmodulin (CaM) at elevated intracellular Ca(2+) levels to produce NO as part of a number of cell signalling pathways. CaM binds to the linker region between the two domains and activates the enzyme by inducing intramolecular electron transfer. Protein-engineering experiments have shown that a series of unusual autoinhibitory inserts found only in the CaM-dependent NOS isoforms control both CaM binding and the structural rearrangement it induces. These lie in the reductase domain of the enzyme and include a 40-amino-acid autoinhibitory loop in the FMN-binding module, a 30-amino-acid extension to the C-terminus and the CaM-binding site itself. The substrate (NADPH) also plays an important role in defining the CaM-dependence of the reductase domain by inducing a tight conformational lock in the absence of CaM. Both the substrate and the conformational lock appear to be released on CaM binding; the resultant domain mobility leads to activation.

Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase.

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

Theoretical Study of 9-β-d-Erythrofuranosyladenine and Corresponding Carbocyclic Analogues. Evidence for a Base-Activated Conformational Lock

The conformational surfaces of three nucleoside analogues have been investigated computationally, where an adenine is attached to a diol of tetrahydrofuran, a diol of cyclopentane, and a diol of cyclopentene. In each system, the lowest-energy conformer displays a conformational lock into the south position by an internal hydrogen bond between O2‘H of the five-membered ring and the N3 nitrogen of adenine. When aqueous solvation is accounted for by the PCM method, the preference for the locked conformer is diminished. A pseudorotation angle of 9-(trans-2‘,trans-3‘dihydroxycyclopentyl)adenine has been determined to be 176.8° by fitting the measured 3JHH values using PSEUROT which is in good agreement with the calculated value of 169.3°.

Calmodulin Activates Electron Transfer through Neuronal Nitric-oxide Synthase Reductase Domain by Releasing an NADPH-dependent Conformational Lock

Neuronal nitric-oxide synthase (nNOS) is activated by the Ca(2+)-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP(+) release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.

Preorganized ligand arrays based on spirotetrahydrofuranyl motifs. Synthesis of the stereoisomeric 1,8,14-trioxatrispiro[4.1.4.1.4. 1]octadecanes and the contrasting conformational features and ionic binding capacities of these belted ionophores.

The cis,trans trispiro ether 4 is accessible from several synthetic directions as a consequence of a crossover in reaction selectivity when proceeding from nucleophilic attack on the cis dispiro ketone to oxygenation of the alpha,beta-unsaturated ester 17. Its cis,cis isomer 3 was obtained in 17 steps and 14.6% overall yield from 3, 5-dimethoxybenzoic acid by making use of the alicyclic side chain in N as a "conformational lock". Although 4 shows no measurable tendency to complex with alkali metal ions, 3 binds strongly to Li(+) and Na(+) ions, as well as to CH(3)NH(3)(+). Whereas the 3eq conformation is populated in the solid state and in solution, complex formation occurs readily. (13)C NMR studies have defined slow exchange limits as the 2:1 sandwich complex with lithium ion is initially formed and transformed progressively into a 1:1 species upon the addition of more LiClO(4). Only the 2:1 complex with sodium ion is formed during comparable titration with NaClO(4). Association constants, molecular mechanics calculations, and X-ray crystallographic studies provide insight into the binding capacity of this belted tridentate ionophore.

The oxetane conformational lock of paclitaxel: Structural analysis of D-secopaclitaxel

Analysis of the 1H NMR data of paclitaxel in comparison with its oxetane ring-opened analogue D-secopaclitaxel suggests that the oxetane moiety (D-ring) of paclitaxel serves as a conformational lock for the diterpene moiety and the C13 side chain.

Catalytic properties, stability and the structure of the conformational lock in the alkaline phosphatase from Escherichia coli

The activity of oligomeric enzymes is sensitive to the formation and dissociation of the interprotein contacts that make up the conformational lock. The mechanism for this is discussed in this article concerning the alkaline phosphatase (AP) from Escherichia coli. Study of the AP from various source shows that the thermoinactivation curves, obtained under various conditions, have induction periods that may be ascribed to latent structural changes in the conformational lock. The analysis of kinetic curves has allowed us to calculate the minimum number of denaturation stages in the conformational lock ( n=3), i.e., the stable dimer becomes labile and capable of dissociation by the sequential dissociation of two of the three contacts which take part in the conformational lock. Three-dimensional structural analysis of AP from E. coli established that the structure of intersubunit contact is formed by three sites: two identical peripheral sites, formed by loop 1–29 and helix 29–34 (H 29–34) of each subunit and one, located near to the active centers of two subunits. Destruction of two contacts does not effect the catalytic activity but opening the third results in the dissociation of dimers into monomers and loss of catalytic activity. Thus, kinetic calculation is correlated with the results of structural analysis. Oligomers with decreased activity and increased stability were found in solutions of E. coli AP. The structural possibilities for tetramer formation, based on packing of molecules in protein crystals, are discussed.

An approach to (+)-pancratistatin from D-glucose: A conformational lock solves a stereochemical problem

In previous work we discovered that reductive Pd-mediated aryl-enone cyclization of an advanced intermediate resulted in epimerization of a critical stereocenter. Analysis of conformational and steric effects could rationalize the result. Based on that analysis it was anticipated that a conformational lock could suppress the undesired epimerization. Results reported herein confirm that expectation.

N-Substituted 2-(2,6-dinitrophenylamino)propanamides: novel prodrugs that release a primary amine via nitroreduction and intramolecular cyclization.

A series of N-dinitrophenylamino acid amides [(4-CONHZ-2, 6-diNO2Ph)N(R)C(X,Y)CONHPhOMe] were prepared as potential bioreductive prodrugs and reduced radiolytically to study their rates of subsequent intramolecular cyclization. Compounds bearing a free NH group (R = H) underwent rapid cyclization in neutral aqueous buffers (t1/2 < 1 min) following 4-electron reduction, with the generation of a N-hydroxydihydroquinoxalinone and concomitant release of 4-methoxyaniline. Amine release from analogous N-methyl analogues (R = Me) was relatively slow. These results are consistent with intramolecular cyclization of a monohydroxylamine intermediate. The high rates of cyclization/extrusion by these very electron-deficient hydroxylamines suggest that the process is greatly accelerated by the presence of an H-bonding "conformational lock" between the anilino NH group and the adjacent o-nitro group (Kirk and Cohen, 1972). Changes in the phenylcarboxamide side chain or in C-methylation in the linking chain had little effect on the rate of cyclization. The model compounds had 1-electron reduction potentials in the range appropriate for cellular reduction (-373 mV for a measured example) and appeared suitable for development as prodrugs that release amine-based effectors following enzymic or radiolytic reduction. Prodrug examples containing 4-aminoaniline mustard and 5-amino-1-(chloromethyl)benz[e]indoline alkylating units were evaluated but were not activated efficiently by cellular nitroreductases. However, cell killing by the radiation-induced reduction of the latter prodrug was demonstrated.

Influence of remote intramolecular hydrogen bonds on the thermodynamics of molecular recognition of cis-1,3,5-cyclohexanetricar☐ylic acid

Variable temperature binding studies and isothermal titration microcalorimetry were used to probe the thermodynamics of molecular recognition of cis-1,3,5-cyclohexanetricar☐ylic acid by tripodal hosts. Remote intramolecular hydrogen bonds, used to restrict conformationally one of the hosts, exhibit a strong influence on the thermodynamic functions for the binding process ΔH and ΔS, with little effect on ΔG. This suggests that the conformational lock imposed by the intramolecular hydrogen bonds organizes the receptor in a conformation that is not optimal for the binding of the triacid.

Coordinated binding of sugar, calcium, and antibody to macrophage C-type lectin

Mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectin (MMGL) is a type II transmembrane glycoprotein belonging to the C-type lectin family. Our development of monoclonal antibodies led us to discover that a calcium-dependent conformational change is detected by an antibody (termed mAb LOM-11) and that the antibody's binding to the respective site locks the lectin in an active conformation. These findings correspond to the divalent cation-mediated regulatory mechanisms in a family of cell adhesion molecule integrins that have gained much attention. We now provide direct evidence that mAb LOM-11 increases the affinity of the lectin for calcium ions as a mechanism for the conformational lock using a soluble recombinant form of MMGL (rML) produced in bacteria. Furthermore, we discovered by using an enzyme-linked immunosorbent assay that specific monosaccharides induced a binding site for mAb LOM-11 on the immobilized rML under low calcium environments. We also demonstrated that cell surface MMGL on a transfectant cell line underwent a conformational change upon addition of calcium or ligands, as detected by the binding of mAb LOM-11. These properties are reminiscent of ligand-induced binding sites defined for integrins. The present results suggest a possibility that the mAb LOM-11 binding site on the lectin may be a site at which protein-protein interaction helps to fine tune the specificity of the C-type lectins by means of coordinated recognition mechanisms.

Conformations of Anaesthetic Steroids: a1H and13C NMR Study of (2β,3α,5α)-2-[(2R)-Ethyl-4-morpholinyl]-3-hydroxypregnane-11,20-dione and(2β,3α,5α)-2-[(2S)-Ethyl-4-morpholinyl]-3-hydroxypregnane-11,20-dione.

The completely assigned 1H and 13C NMR spectra of the title diastereoisomers in solutions of CDCl3 and DMSO-d6 are reported. NOE experiments show that these molecules adopt specific conformations with no rotation about the C-2—N bond in either solvent. In CDCl3, ring A of the steroid is in a twist-boat conformation and an intramolecular hydrogen bond from the 3α-OH to the morpholine N acts as a conformational lock preventing rotation of the morpholine ring. In DMSO-d6 solutions, ring A of the steroid is in a chair conformation and van der Waals contacts with the 19-methyl group prevent free rotation of the morpholine ring. Molecular mechanics calculations produced four structures that account for the experimental data. © 1997 by John Wiley & Sons, Ltd.

ChemInform Abstract: Nucleosides and Nucleotides. Part 115. Synthesis of 3‐Alkyl‐3‐deazainosines via Palladium‐Catalyzed Intramolecular Cyclization: A New Conformational Lock with the Alkyl Group at the 3‐Position of the 3‐Deazainosine in anti‐Conformation.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

2′- C-, 3′- C- and 5′- C-methylsangivamycins: conformational lock with the methyl group

Introduction of the methyl groups into the appropriate positions of the ribose moiety in sangivamycin ( 1) induced the deviation of the conformer population which could be predicted by the molecular mechanics calculations.

New trialkylsilyl enol ether chemistry. Synthesis of the benzomorphanone core structure using a stereoelectronic conformational lock

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNew trialkylsilyl enol ether chemistry. Synthesis of the benzomorphanone core structure using a stereoelectronic conformational lockPhilip Magnus and Iain ColdhamCite this: J. Am. Chem. Soc. 1991, 113, 2, 672–673Publication Date (Print):January 1, 1991Publication History Published online1 May 2002Published inissue 1 January 1991https://doi.org/10.1021/ja00002a044RIGHTS & PERMISSIONSArticle Views312Altmetric-Citations31LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (243 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

The conformation of 4-phenylcyclohexanone in solution by 1H- and 13C-Lanthanide induced shift (L.I.S.) analysis : Evidence in favour of phenyl over t-butyl as a conformational lock

The degree of pucker of the cyclohexanone ring in 4-phenylcyclohexanone has been refined by L.I.S. analysis. The results indicate puckering intermediate between that in cyclohexanone and 4-t-butylcyclohexanone, suggesting that phenyl may be preferable to t-butyl as a locking group in conformational analysis. The Yb (fod)3 shift reagent, although binding primarily at the carbonyl oxygen atom, also complexes weakly with the phenyl group.